Classical swine fever (CSF) is one of the most important diseases of pigs. Although prophylactic vaccination is banned within the European Union, emergency vaccination, allowing differentiation of vaccinated from infected animals, is an option for disease control. Up to now, these strategies are based on antibody detection. In this context, conventional modified live vaccines are not suitable. A promising perspective could be genetic differentiation of vaccinated from infected animals where field virus strains are differentiated from vaccine viruses by sequence differences. This concept could also be used with marker vaccines. To this end, a set of real-time reverse transcription-polymerase chain reaction (RT-PCR) assays was developed and validated. Specific primers and probes were designed for detection of the C-strain "Riems" vaccine virus or the chimeric marker vaccine candidate CP7_E2alf. A heterologous internal positive control was also included. The assays were then multiplexed to detect simultaneously either CSF field virus, C-strain "Riems", and the internal control or CSF field virus, CP7_E2alf, and the internal control. To validate both systems, samples from vaccination/challenge trials were tested. Only samples from vaccinated animals were found to be positive, while all samples from wild type virus-infected animals and a broad test panel of different pestiviruses were negative. Field application of the "C-strain Riems" specific assay was proven with wild boar samples from surveillance programs in Germany and France. In conclusion, ready-to-use RT-PCR sets are presented as reliable tools for genetic differentiation of vaccinated from infected animals for CSFV eradication strategies.